EP3433271A1 - Methods of detecting apobec3 expression and predicting clinical outcomes - Google Patents
Methods of detecting apobec3 expression and predicting clinical outcomesInfo
- Publication number
- EP3433271A1 EP3433271A1 EP17771143.9A EP17771143A EP3433271A1 EP 3433271 A1 EP3433271 A1 EP 3433271A1 EP 17771143 A EP17771143 A EP 17771143A EP 3433271 A1 EP3433271 A1 EP 3433271A1
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- Prior art keywords
- apobec3
- expression
- level
- apobec3g
- antibody
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/087—Herpes simplex virus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
Definitions
- this disclosure describes a method that includes: detecting a level of
- the method further includes providing treatment to the subject based upon the prognosis.
- this disclosure a method for making an anti-APOBEC3 antibody / APOBEC3 complex.
- the method includes: providing a sample including APOBEC3; and contacting the sample with an anti-APOBEC3 antibody under conditions that permit the binding of the anti-APOBEC3 antibody to APOBEC3 to yield the anti-APOBEC3 antibody / APOBEC3 complex.
- the sample can be from a subject having a cancer or a tumor.
- this disclosure describes a kit for determining a level of
- the kit includes reagents for determining the level of expression of APOBEC3 in the sample; and instructions for how to use the reagents.
- this disclosure describes a method including detecting APOBEC3 in a sample from a subject and detecting the level of expression of a T cell marker in the sample.
- the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
- FIG. 1 shows correlations between APOBEC3 expression and T cell markers in high- grade serous ovarian carcinoma (HGSOC).
- HOSOC high- grade serous ovarian carcinoma
- Dot plots illustrating correlations between APOBEC3G (panels A-F) or APOBEC3B (panels G-L) expression and the indicated T cell marker (n 354).
- mRNA expression was determined using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and normalized to the housekeeping gene TBP.
- FIG. 2 shows immunohistochemistry and immunofluorescence of T cell markers in HGSOC.
- the dotted box in the 40x hematoxylin images indicates the approximate location of the subsequent panels at lOOx magnification.
- FIG. 3 shows clinical correlates of T cell marker expression in HGSOC.
- FIG. 4 shows correlations between APOBEC expression and immune cell markers across 22 cancer types.
- Heatmap of Spearman's correlation coefficients calculated from the comparison of the T cell marker CD3D (top panel) or the B cell marker CD20 (bottom panel) with the indicated APOBEC family member. Expression levels were determined using TCGA RNAseq data (see Table 5 for the long form for each tumor abbreviation).
- FIG. 5 shows validation of RT-qPCR assays.
- Panel A Histograms reporting mRNA levels of CD3D, CD4, and CD8A in peripheral blood mononuclear cells and CD4+ T cells isolated from the same donor. mRNA levels were quantified by RT-qPCR and represent the average of triplicate reactions. Error bars denote standard deviation (SD).
- Panel B Histograms reporting mRNA levels of CD3D, CD4, and CD8A in peripheral blood mononuclear cells and CD4+ T cells isolated from the same donor. mRNA levels were quantified by RT-qPCR and represent the average of triplicate reactions. Error bars denote standard deviation (SD).
- SD standard deviation
- FIG. 6 shows clinical correlates of APOBEC3G expression in two independent cohorts of HGSOC. Samples were split at the median APOBEC3G expression value with the light gray line representing tumors with high mRNA levels and the dark gray line
- compositions, kits, and methods of using APOBEC3 expression as a predictive biomarker for anti-cancer responses and clinical outcomes are needed.
- this disclosure describes a method of determining a prognosis of a subject having a cancer. In some cases, the subject may be treated based on the prognosis.
- the disclosure further describes a kit for determining the level of APOBEC3 in a sample of a subject, and a method for making an anti-APOBEC3 antibody / APOBEC3 complex.
- Ovarian cancer is the deadliest gynecological malignancy worldwide.
- the most common type of ovarian cancer, high-grade serous ovarian carcinoma (HGSOC) accounts for over 60% of cases, and is the most aggressive reproductive track malignancy.
- HGSOC high-grade serous ovarian carcinoma
- T cell infiltration has been identified as a favorable prognostic factor for HGSOC (Knutson et al. Cancer Immunol Immunother. 2015;64: 1495-504; Nielsen et al. Clin Cancer Res. 2012; 18:3281-92; Zhang et al. N Engl J Med. 2003;348:203-13; Sato et al.
- the APOBEC enzymes are an 11 -member family of zinc-coordinating enzymes that convert cytosines to uracils (C-to-U) in ssDNA.
- the APOBEC enzymes include APOBEC 1 (isoform a, NP_001291495.1; isoform b, NP_005880.2), APOBEC2 (NP_006780.1), APOBEC3A (isoform a, NP_663745.1; isoform b, NP_001257335.1), APOBEC3B
- NP 004891.4 APOBEC3C
- APOBEC3D NP 689639.2
- APOBEC3F NP_660341.2
- APOBEC3G isoform 1, NP_068594.1; isoform 2, NP_001336365.1;
- AID activation-induced deaminase
- the enzymatic activity of specific family members including, for example, AID, is essential for both adaptive and innate immune responses. AID plays a role in antibody diversification through somatic hypermutation and class switch recombination in B cells.
- Other well-studied family members include APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H.
- APOBEC3 subfamily members have also been implicated in restricting the replication of many other DNA-based parasites including transposable elements. Most APOBEC family members are expressed broadly and constitutively, but several family members can also be further upregulated by specific conditions.
- APOBEC3A can be upregulated by interferon-a; APOBEC3B can be upregulated by non-canonical NF- ⁇ activation and HPV infection; and APOBEC3C, APOBEC3D, APOBEC3F, APOBEC 3G, and APOBEC 3H can be upregulated by the combination of T cell activation and HIV-1 infection.
- APOBEC3 enzymes have been shown to have physiologic roles in protecting cells from endogenous and exogenous DNA-based pathogens, their dysregulation has also been linked to cancer. For instance, AID has been linked to various B cell malignancies and APOBEC3B is overexpressed and a significant source of mutation in breast, ovarian, and several other cancers.
- APOBEC3B deaminates cytosines in genomic DNA to produce promutagenic uracil lesions, which can result in mutations if they are not repaired faithfully.
- APOBEC3B is overexpressed and its mutation signature is
- APOBEC3B In addition to APOBEC3B, related subfamily members have also been implicated in cancer mutagenesis to varying degrees. Because most APOBEC family members are expressed in many normal cell types and tissues, a major confounding factor in quantification of APOBEC expression levels in tumors is cellular heterogeneity due to surrounding normal tissue and immune cell infiltration. To address these issues, the disclosure describes quantification of APOBEC expression in a large cohort of HGSOC patients and exploration of the correlation of APOBEC expression levels with immune cell infiltration. A strong positive correlation was identified between APOBEC3G expression and markers of T cell infiltration, and co-expression was confirmed by immunohistochemical and
- RNAseq The Cancer Genome Atlas
- This disclosure describes the unanticipated association between high levels of APOBEC3G expression and cytotoxic T lymphocyte (CTL) activation in HGSOC.
- CTL cytotoxic T lymphocyte
- This disclosure further describes the surprising identification of APOBEC3G as a new candidate biomarker for activated T lymphocyte infiltration in HGSOC and improved patient outcomes. This role for APOBEC3G is unexpected in part because prior work has shown that
- APOBEC3G is expressed broadly, constitutively, and in some instances inducibly (Refsland et al. Nucleic Acids Res. 2010;38:4274-8; Hultquist et al. Journal of Virology.
- FIG. 1 A-E The analysis of a cohort of 354 HGSOC patients also identified a strong correlation between APOBEC3G and several markers of T cell infiltration. These results were validated at the protein level by immunohistochemistry and immunofluorescent staining of independent HGSOC tumor samples (representative image sets in FIG. 2). Clinical data revealed that APOBEC3G also associates with improved outcomes in a large HGSOC cohort as well as in two additional independent ovarian cancer cohorts (Table 2, FIG. 3, and FIG. 6). Finally, a global analysis of gene expression in 22 cancer types identified a similar correlation for two additional APOBEC3 genes, APOBEC3D and APOBEC3H, and a marker of T cells, CD3D (FIG. 4). Together, these data suggest that APOBEC3D, APOBEC3G, and APOBEC3H expression levels in tumor infiltrating T lymphocytes may be a predictive biomarker for strong anti-cancer T cell responses and improved HGSOC outcomes.
- APOBEC3B and AID contribute to cancer genome mutagenesis.
- APOBEC3B is thought to mutate the genome of several different cancer types, including breast, lung, bladder, cervical, head/neck, and ovarian (Burns et al. Nature. 2013;494:366-70; Leonard et al. Cancer Research. 2013;73 :7222-31; Burns et al. Nat Genet. 2013;45:977-83; Roberts et al. Nat Genet. 2013;45:970-6; Alexandrov et al. Nature. 2013;500:415-21).
- the carcinogenic effect of AID appears more limited, as its characteristic deamination signature has only been found in certain types of B cell leukemias and lymphomas (Robbiani et al. Annu Rev Pathol.
- AID expression is constrained to B lineage cell types is consistent with data showing that AID mRNA expression correlates with CD20 mRNA expression in several solid tumor types (FIG. 4 bottom heatmap).
- APOBEC3B and AID several other APOBEC family members have also been implicated in carcinogenesis.
- APOBEC3G may drive hepatocellular carcinoma tumorigenesis.
- the idea that APOBEC3G drives tumorigenesis is, however, difficult to reconcile with the observation that APOBEC '3G mRNA expression in HGSOC and other tumor types correlates with the expression of activated T lymphocyte markers. Moreover, at least for HGSOC, this strong correlation could be validated visually at the protein level (FIGs. 1, 2, and 4).
- TNBC triple negative breast cancer
- CD3D was substantially diminished as compared to APOBEC3D, APOBEC3G, and
- APOBEC3H (FIG. 4). This result is surprising because APOBEC3F is thought to be broadly expressed and to play an equally important role as APOBEC3D, APOBEC3G, and
- APOBEC3H in HIV-1 restriction in CD4-positive T lymphocytes, and because APOBEC3F is expressed at comparable levels to APOBEC3D and at higher levels than APOBEC3H in human primary CD4+ T cells.
- the data in Example 1 suggest that APOBEC3F may be under- and/or heterogeneously-expressed in T cells associated with the tumor
- Example 1 further indicate that much of the expression of several APOBEC family members in cancer is likely due to T and B cell infiltration.
- this disclosure describes a method of determining a prognosis of a subject having a cancer.
- the method further includes treating the patient based on the prognosis.
- the cancer is a primary tumor.
- the cancer is metastatic (i.e., disseminated beyond the site of the primary tumor).
- a cancer is a solid tumor.
- the cancer is a blood cancer (for example, a leukemia or a lymphoma).
- the cancer can involve any tissue or organ, such as bone, brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, uterus, ovary, or blood.
- the cancer can be a bone cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, cancer of the larynx, lung cancer, pancreatic cancer, prostate cancer, skin cancer, cancer of the spine, stomach cancer, uterine cancer, or a blood cancer.
- the cancer can be a carcinoma.
- the cancer includes one of the cancers listed in Table 5.
- the cancer includes bladder urothelial carcinoma, acute myeloid leukemia, brain lower grade glioma, glioblastoma multiforme, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, prostate adenocarcinoma, breast invasive carcinoma, lung adenocarcinoma, thyroid carcinoma, skin cutaneous melanoma, esophageal carcinoma, lung squamous cell carcinoma, uterine corpus endometrial carcinoma, pancreatic adenocarcinoma, stomach adenocarcinoma, testicular germ cell tumor, ovarian carcinoma, ovarian serous cystadenocarcinoma, kidney renal papillary cell carcinoma, or high-grade serious ovarian cancer (HGSOC).
- bladder urothelial carcinoma acute myeloid leukemia, brain lower grade glioma, glioblastoma multiforme, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, prostate adenocarcinoma, breast invasive carcinoma,
- the method of determining a prognosis of the subject includes detecting the level of APOBEC3 expression in the sample from the subject; and determining the prognosis of the subject based upon the level of APOBEC3 expression.
- the method can include obtaining a sample from the subject.
- the sample is preferably derived from a tumor.
- APOBEC3 includes one or more of the APOBEC3 family members: APOBEC3A, APOBEC3B, APOBEC3C,
- the APOBEC3 includes APOBEC3D, APOBEC3G, and/or APOBEC3H. In some embodiments, the APOBEC3 preferably includes APOBEC3G.
- detecting the level of APOBEC3 expression includes detecting the level of expression of an RNA or a protein of one or more members of the APOBEC3 family. In some embodiments, detecting the level of APOBEC3 expression preferably includes detecting the RNA level of APOBEC3D, APOBEC3G, and/or APOBEC3H or detecting the protein level of APOBEC3D, APOBEC3G, and/or APOBEC3H.
- detecting the level of APOBEC3 expression preferably includes detecting the RNA level of APOBEC3D, APOBEC3G, and/or APOBEC3H or detecting the protein level of APOBEC3D, APOBEC3G, and/or APOBEC3H.
- the sample is a tissue sample including, for example, a tumor sample.
- the sample can be obtained from the subject using any suitable method.
- a biopsy can be used to obtain the sample including, for example, a punch biopsy or a core needle biopsy.
- the sample includes a cell derived from a tumor.
- a cell derived from a tumor can include a tumor cell and/or a tumor infiltrating cell including, for example, a lymphocyte.
- the sample includes a tumor cell.
- the sample includes a tumor-infiltrating cell including, for example, a lymphocyte and/or a T cell.
- the sample includes a tissue section.
- the tissue sample includes a cell including at least one of the cancers listed in Table 5.
- the tissue sample includes a cell including at least one of a bladder urothelial carcinoma, an acute myeloid leukemia, a brain lower grade glioma, a glioblastoma multiforme, a kidney renal clear cell carcinoma, a liver hepatocellular carcinoma, a prostate adenocarcinoma, a breast invasive carcinoma, a lung adenocarcinoma, a thyroid carcinoma, a skin cutaneous melanoma, an esophageal carcinoma, a lung squamous cell carcinoma, an uterine corpus endometrial carcinoma, a pancreatic adenocarcinoma, a stomach adenocarcinoma, a testicular germ cell tumor, an ovarian carcinoma, an ovarian serous cystadenocarcinoma, a kidney renal papillary cell carcinoma, or a high-grade serious
- the subject can be a mammalian subject. In some embodiments, the subject can be a mammalian subject. In some
- the subject can be a primate. In some embodiments, the subject can be a human subject. In some embodiments, the subject can be a cancer patient.
- detecting the level of APOBEC3 expression can include RNA- level detection and/or quantification of APOBEC3 expression. In some embodiments, detecting the level of APOBEC3 expression includes RNA-level detection of a nucleotide sequence including an APOBEC3D RNA, an APOBEC3G RNA, or an APOBEC3H RNA.
- APOBEC3 expression can be detected by any suitable method including, for example, by using quantitative reverse transcription polymerase chain reaction (RT-qPCR).
- the level of APOBEC3 expression can be detected by, for example, Northern blotting, microarray detection, etc.
- APOBEC3 expression can be detected via a cDNA intermediate through, for example, RTqPCR and RNAseq.
- a primer can include SEQ ID NO: l and/or SEQ ID NO: 13.
- detecting the level of APOBEC3 expression can include protein level detection and/or quantification.
- Protein expression may be determined by any suitable method including, for example, antibody-based methods and non-antibody based methods.
- Antibody -based methods can include, for example, western blot, flow cytometry, immunofluorescence, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, etc.
- Non-antibody based methods can include, for example, protein array, interactor binding (e.g., Vif), enzyme fragment complementation, MALDI-TOF, mass spectrometry, amino acid analysis, etc.
- detecting the level of APOBEC3 expression can include using an anti-APOBEC3 antibody.
- Any suitable anti-APOBEC3 antibody can be used.
- the anti- APOBEC3 antibody can include, for example, an anti-APOBEC3D, an anti-APOBEC3G, or an anti-APOBEC3H antibody.
- the anti-APOBEC3 antibody can include an antibody produced by a hybridoma cell line (including those described in copending application number PCT/US2016/040011, which is herein incorporated by reference), or an antibody produced by phage display technologies.
- the anti-APOBEC3 antibody can include a monoclonal antibody produced by a hybridoma cell line. In some embodiments the anti-APOBEC3 antibody can include a monoclonal antibody produced recombinantly after isolation from a hybridoma line. For example, heavy and light chains may be cloned from a hybridoma line. In some embodiments, the anti-APOBEC3 antibody includes any anti-APOBEC3D, anti- APOBEC3G, or anti-APOBEC3H antibody that works in immunohistochemistry procedures.
- an anti-APOBEC3G antibody can include HPA001812 (Sigma-Aldrich, St. Louis, MO). In some embodiments the anti-APOBEC3G antibody can include a monoclonal antibody produced by hybridoma cell line 5211-110-19 (described in co-pending application number PCT/US2016/040011, which is herein incorporated by reference). In some embodiments the anti-APOBEC3G antibody can include a monoclonal antibody produced by hybridoma cell line 5210-87-13 (also described in co-pending application number PCT/US2016/040011).
- each of these rabbit monoclonal antibodies recognizes APOBEC3G, APOBEC3A, and APOBEC3B due to unavoidable homology, under certain conditions, the antibodies only detect APOBEC3G (e.g., during immunohistochemistry) because APOBEC3 A is not expressed in T lymphocytes.
- a monoclonal antibody produced by hybridoma cell line 5211-110-19 did not recognize endogenous APOBEC3B during the immunohistochemistry procedures described herein.
- the method further comprises detecting an anti-APOBEC3 antibody.
- the anti-APOBEC3 antibody can be detected using a detection reagent including, for example, a secondary antibody, an enzyme, a fluorophore, a radioactive label, or a luminophore.
- the anti-APOBEC3 antibody may be conjugated to a detection reagent.
- the anti-APOBEC3 antibody may be conjugated to a chip, a biochip, a bead, a microarray, and/or other high throughput solution.
- the method further comprises comparing the level of expression of APOBEC3 in the sample to a control level of expression of APOBEC3.
- a "control level of expression" of APOBEC3 may be the level of APOBEC3 expression in non-transformed tissue of the same subject; the level of a APOBEC3 expression in tissue from a control subject; or a known value of APOBEC3 expression based on the level of expression in a tissue in a pool of control subjects.
- a control subject may be a subject without a cancer and/or a tumor.
- a control subject may be a subject with a similar cancer and/or tumor and a known prognosis.
- an increased level of APOBEC3 in the sample compared to a control level of expression of an APOBEC3 may be associated with a good prognostic outcome.
- an increased level of APOBEC3G in the sample compared to a control level of expression of APOBEC3G may be associated with a good prognostic outcome.
- an increased level of APOBEC3D in the sample compared to a control level of expression of APOBEC3D may be associated with a good prognostic outcome.
- an increased level of APOBEC3H in the sample compared to a control level of expression of APOBEC3H may be associated with a good prognostic outcome.
- an increased level of a particular isoform or splice variant of APOBEC3 in a sample compared to a control level of expression of the corresponding isoform or splice variant of APOBEC3 may be associated with a good prognostic outcome.
- NP_068594.1of APOBEC3G may be associated with a good prognostic outcome; an increased level of isoform 2, NP 001336365.1 of APOBEC3G in the sample compared to a control level of expression of isoform 2, NP 001336365.1 of APOBEC3G may be associated with a good prognostic outcome; an increased level of isoform 3, NP 001336366.1 of APOBEC3G in the sample compared to a control level of expression of isoform 3,
- NP 001336366.1 of APOBEC3G may be associated with a good prognostic outcome; and/or an increased level of isoform 4, NP 001336367.1 of APOBEC3G in the sample compared to a control level of expression of isoform 4, P 001336367.1 of APOBEC3G may be associated with a good prognostic outcome.
- the method further comprises determining the expression of a T cell marker in the sample. In some embodiments, determining the expression of a T cell marker includes determining the level of expression of the T cell marker.
- the T cell marker can include, for example, CD3 (delta chain isoform A, NP_000723.1; delta chain isoform B, NP_001035741.1; zeta chain isoform 1, P_932170.1; zeta chain isoform 2, NP_000725.1; gamma chain, NP_000064.1; epsilon chain
- the T cell marker is a marker of cytotoxic T cell activation.
- determining the expression of a T cell marker comprises by detecting the transcript of a gene that encodes the T cell marker.
- an increased level of at least one T cell marker in the sample compared to a control level of expression of the T cell marker may be associated with a good prognostic outcome.
- an increased level of APOBEC3 and an increased level of at least one T cell marker in a sample compared to a control level of expression of APOBEC3 and a control level of expression of the T cell marker may be associated with a good prognostic outcome.
- determining the prognosis of the subject based upon the level of APOBEC3 expression can include comparing the level of expression of APOBEC3 in the sample to the level of a APOBEC3 expression in non-transformed tissue of the same subject; the level of a APOBEC3 expression in tissue from a control subject; or a known value of APOBEC3 expression based on the level of expression in a tissue in a pool of control subjects.
- a control subject may be a subject without a cancer and/or a tumor.
- a control subject may be a subject with a similar cancer and/or tumor and a known prognosis.
- determining the prognosis of the subject includes predicting a clinical outcome including, for example, the length of progression free survival (PFS) and/or overall survival (OS).
- PFS progression free survival
- OS overall survival
- determining the prognosis of the subject can include determining whether the subject is likely to be responsive to a therapy. In some
- determining the prognosis of the subject can include determining the subject's responsiveness to a therapy.
- the therapy can include any cancer therapy alone or in combination including, for example, immunotherapy, chemotherapy, surgery, and/or radiation.
- APOBEC3 expression is especially likely to be predictive of therapeutic success of therapies that are dependent on T cell infiltration including, for example, T cell-based
- APOBEC3 family members including APOBEC3D, APOBEC3G, and/or APOBEC3H
- the expression and/or level of expression of certain APOBEC3 family members may predict the number of T cells available to affect anticancer responses in the tumor.
- the expression and/or level of expression of certain APOBEC3 family members may predict the number of T cells available to affect anticancer responses in the tumor.
- APOBEC3 family members may further predict the number of activated or non-inhibited T cells available to affect anti-cancer responses in the tumor.
- the method further includes treating the patient.
- the treatment chosen may be based on the prognosis.
- the treatment may include, for example,
- an immunotherapy is a T cell-based immunotherapy including, for example, a chimeric antigen receptor (CAR) T cell.
- an immunotherapy can be a checkpoint inhibitor including, for example, anti-CTLA4 and/or an anti-PDl agent.
- chemotherapy can include the use of a chemotherapeutic agent including, for example, carboplatin, paclitaxel, doxorubicin, gemcitabine, Topotecan, and/or a Poly (ADP- ribose) polymerase (PARP) inhibitor.
- a chemotherapeutic agent including, for example, carboplatin, paclitaxel, doxorubicin, gemcitabine, Topotecan, and/or a Poly (ADP- ribose) polymerase (PARP) inhibitor.
- PARP Poly (ADP- ribose) polymerase
- the kit includes reagents for determining the level of expression of APOBEC3 in a sample and instructions for how to use the reagents. In some embodiments the kit includes instructions for how to use the reagents. In some embodiments, the instructions are FDA-approved.
- the sample is a tissue sample.
- the sample can be obtained from the subject using any suitable method.
- a biopsy can be used to obtain the sample including, for example, a punch biopsy or a core needle biopsy.
- the sample includes a sample from a cancer.
- a sample from a cancer can include a tumor cell and/or a tumor infiltrating cell including, for example, a lymphocyte.
- the sample includes a tumor cell.
- the sample includes a tissue section.
- the tissue sample includes a cell including at least one of the cancers listed in Table 5.
- the tissue sample includes a cell including at least one of a bladder urothelial carcinoma, an acute myeloid leukemia, a brain lower grade glioma, a glioblastoma multiforme, a kidney renal clear cell carcinoma, a liver hepatocellular carcinoma, a prostate adenocarcinoma, a breast invasive carcinoma, a lung adenocarcinoma, a thyroid carcinoma, a skin cutaneous melanoma, an esophageal carcinoma, a lung squamous cell carcinoma, an uterine corpus endometrial carcinoma, a pancreatic adenocarcinoma, a stomach adenocarcinoma, a testicular germ cell tumor, an ovarian carcinoma, an ovarian serous cystadenocarcinoma, a kidney renal papillary cell carcinoma, or a high-grade serious
- the subject can be a mammalian subject. In some embodiments, the subject can be a mammalian subject. In some
- the subject can be a primate. In some embodiments, the subject can be a human subject. In some embodiments, the subject can be a cancer patient.
- the cancer is a primary tumor. In some embodiments, the cancer is metastatic (i.e., disseminated beyond the site of the primary tumor). In some embodiments, a cancer is a solid tumor. In some embodiments, the cancer is a blood cancer (for example, a leukemia or a lymphoma).
- the cancer can involve any tissue or organ, such as bone, brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, uterus, ovary, or blood.
- the cancer can be a bone cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, cancer of the larynx, lung cancer, pancreatic cancer, prostate cancer, skin cancer, cancer of the spine, stomach cancer, uterine cancer, or a blood cancer.
- the cancer can be a carcinoma.
- the cancer includes one of the cancers listed in Table 5.
- the cancer includes bladder urothelial carcinoma, acute myeloid leukemia, brain lower grade glioma, glioblastoma multiforme, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, prostate adenocarcinoma, breast invasive carcinoma, lung adenocarcinoma, thyroid carcinoma, skin cutaneous melanoma, esophageal carcinoma, lung squamous cell carcinoma, uterine corpus endometrial carcinoma, pancreatic adenocarcinoma, stomach adenocarcinoma, testicular germ cell tumor, ovarian carcinoma, ovarian serous cystadenocarcinoma, kidney renal papillary cell carcinoma, or high-grade serious ovarian cancer (HGSOC).
- bladder urothelial carcinoma acute myeloid leukemia, brain lower grade glioma, glioblastoma multiforme, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, prostate adenocarcinoma, breast invasive carcinoma,
- detecting the level of APOBEC3 expression includes detecting the level of expression of an RNA or a protein of one or more members of the APOBEC3 family. In some embodiments, detecting the level of APOBEC3 expression preferably includes detecting an RNA level of APOBEC3D, APOBEC3G, and/or APOBEC3H or detecting a protein level of APOBEC3D, APOBEC3G, and/or APOBEC3H. In some embodiments, the APOBEC3 preferably includes APOBEC3G.
- determining the level of expression of APOBEC3 can include RNA-level detection and/or quantification of APOBEC3 expression.
- detecting the level of APOBEC3 expression includes RNA-level detection of a transcript including APOBEC3D, APOBEC3G, or APOBEC3H.
- APOBEC3 expression can be detected by any suitable method including, for example, by using quantitative reverse transcription polymerase chain reaction (RT-qPCR).
- RT-qPCR quantitative reverse transcription polymerase chain reaction
- the level of APOBEC3 expression can be detected by, for example, Northern blotting, microarray detection.
- APOBEC3 expression can be detected via a cDNA intermediate through, for example, RTqPCR and RNAseq.
- reagents for determining the level of expression of APOBEC3G include reverse transcriptase and/or a pair of primers which are specific to separate regions of APOBEC3G.
- the primers comprise SEQ ID NO: l and/or SEQ ID NO: 13.
- reagents for determining the level of expression of APOBEC3 include reagents for protein level detection and/or quantification.
- Protein expression may be determined by any suitable method including, for example, antibody -based methods and non- antibody based methods.
- Antibody -based methods can include, for example, western blot, flow cytometry, immunofluorescence, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, etc.
- Non-antibody based methods can include, for example, protein array, interactor binding (e.g., Vif), enzyme fragment complementation, MALDI-TOF, mass spectrometry, amino acid analysis, etc.
- reagents for determining the level of expression of APOBEC3 include an anti-APOBEC3 antibody.
- Any suitable anti-APOBEC3 antibody can be used.
- the anti-APOBEC3 antibody can include, for example, an anti-APOBEC3D, an anti- APOBEC3G, or an anti-APOBEC3H antibody.
- the anti-APOBEC3 antibody can include an antibody produced by a hybridoma cell line (including those described in co-pending application number PCT/US2016/040011, which is herein incorporated by reference), or an antibody produced by phage display technologies.
- the anti-APOBEC3 antibody can include a monoclonal antibody produced by a hybridoma cell line. In some embodiments the anti-APOBEC3 antibody can include a monoclonal antibody produced recombinantly after isolation from a hybridoma line. For example, heavy and light chains may be cloned from a hybridoma line. In some embodiments, the anti-APOBEC3 antibody includes any anti-APOBEC3D, anti- APOBEC3G, or anti-APOBEC3H antibody that works in immunohistochemistry procedures.
- an anti-APOBEC3G antibody can include HPA001812
- the anti-APOBEC3G antibody can include a monoclonal antibody produced by hybridoma cell line 5211-110-19 (described in co-pending application number PCT/US2016/040011, which is herein incorporated by reference). In some embodiments the anti-APOBEC3G antibody can include a monoclonal antibody produced by hybridoma cell line 5210-87-13 (also described in co-pending application number PCT/US2016/040011).
- the reagents can include a detection reagent for detecting an anti-APOBEC3 antibody including, for example, a secondary antibody, an enzyme, a fluorophore, a radioactive label, or a luminophore.
- the anti- APOBEC3 antibody may be conjugated to a detection reagent. In some embodiments, the anti-APOBEC3 antibody may be conjugated to a chip, a biochip, a bead, a microarray, and/or other high throughput solution.
- the kit may further include reagents for comparing the level of expression of APOBEC3 in the sample to a control level of expression of APOBEC3.
- a "control level of expression" of APOBEC3 may be the level of APOBEC3 expression in non-transformed tissue of the same subject; the level of a APOBEC3 expression in tissue from a control subject; or a known value of APOBEC3 expression based on the level of expression in a tissue from a pool of control subjects.
- a control subject may be a subject without a cancer and/or a tumor.
- a control subject may be a subject with a similar cancer and/or tumor and a known prognosis.
- the kit further includes reagents for determining the level of expression of a T cell marker.
- the reagents for determining the level of expression of a T cell marker can include an antibody that is specific to a T cell marker.
- the T cell marker can include, for example, CD3, CD3D, CD4, CD8, CD8A, GZMB, PRFl, etc.
- the T cell marker can be a marker of cytotoxic T cell activation.
- the reagents for determining the level of expression of a T cell marker can include a primer or primers that are specific to a T cell marker
- determining the level of APOBEC3 comprises detecting APOBEC3 expression in tumor-infiltrating T cells.
- APOBEC3 expression in tumor- infiltrating T cells may be detected by an suitable method including, for example, by immunohistochemistry.
- the kit may include antibodies to detect APOBEC3 and one or more T cell markers in a tissue section, and instructions to determine the co-localization of APOBEC3G with a T cell marker via
- the kit may include antibodies to detect APOBEC3 and one or more T cell markers , and instructions to determine the co-expression of APOBEC3G with a T cell marker on a single cell via flow cytometry. In some embodiments, the kit may further include instruction for determining the prognosis of the subject based upon the level of APOBEC3 expression.
- Such instructions can include comparing the level of expression of APOBEC3 in the sample to a control level of expression of APOBEC3; to the level of a APOBEC3 expression in non-transformed tissue of the same subject; to the level of a APOBEC3 expression in tissue from a control subject; or to a known value of APOBEC3 expression based on the level of expression in a tissue in a pool of control subjects.
- this disclosure describes a method for making an anti-APOBEC3 antibody / APOBEC3 complex.
- the method includes providing a sample comprising APOBEC3; and contacting the sample with an anti-APOBEC3 antibody under conditions that permit the binding of the anti-APOBEC3 antibody to APOBEC3 to yield the anti-APOBEC3 antibody / APOBEC3 complex.
- the sample can be from a subject having a cancer or a tumor. In some embodiments, the sample can be from the cancer or tumor.
- the APOBEC3 is one or more members of the APOBEC3 family. In some embodiments, the APOBEC3 preferably includes APOBEC3D,
- the APOBEC3 preferably includes APOBEC3G.
- the anti-APOBEC3 antibody includes, for example, an anti-
- APOBEC3D an anti-APOBEC3G, or an anti-APOBEC3H antibody.
- Any suitable anti- APOBEC3 antibody can be used.
- the antibody can include an antibody produced by a hybridoma cell line (including those described in co-pending application number PCT/US2016/040011, which is herein incorporated by reference), or an antibody produced by phage display technologies.
- an anti-APOBEC3G antibody can include HPA001812 (Sigma-Aldrich, St. Louis, MO).
- the anti-APOBEC3G antibody includes a monoclonal antibody produced by hybridoma cell line 5211-110-19 (described in co-pending application number PCT/US2016/040011, which is herein incorporated by reference).
- the anti-APOBEC3G antibody includes a monoclonal antibody produced by hybridoma cell line 5210-87-13 (described in co-pending application number PCT/US2016/040011).
- RNA samples from 354 high-grade serous ovarian cancer (HGSOC) cases at the Mayo Clinic were selected based on histology, grade, stage, and availability of clinical outcome data (IRB #13-002487).
- TRIzol based RNA extractions were performed.
- cDNA was synthesized in triplicate using Transcriptor Reverse Transcriptase (F. Hoffmann-La Roche, Basel, Switzerland) and reverse transcription quantitative PCR (RT-qPCR) for APOBEC3G, APOBEC3B, CD3D, CD4, CD8A, GZMB, PRF1, RNF128, and TBP was performed using the primer and probe combinations listed in Table 3 (validation in FIG. 5).
- Correlations between APOBEC3G, APOBEC3B, and the various T cell markers were determined using Spearman's correlation. Spearman's correlation coefficient (r s ) and p-values are reported.
- Immunohistochemistry (IHC) for CD3, CD4, CD8, and APOBEC3G was performed on 7 paraffin-embedded primary HGSOC specimens obtained from patients who underwent debulking surgery at Ghent University Hospital. The tissues were fixed in a 4%
- mouse monoclonal anti-CD3 (clone F7.2.38, dilution 1/10, Dako, Carpinteria, CA)
- rabbit monoclonal anti-CD4 (clone SP35, dilution 1/25, Cell Marque Corporation, Rocklin, CA)
- mouse monoclonal anti-CD8 (clone C8/144B, no dilution, Dako, Carpinteria, CA)
- a rabbit monoclonal anti-APOBEC3G (clone 5211-110-19 (described in co-pending application number PCT/US2016/040011, which is herein incorporated by reference), dilution 1/50).
- this rabbit monoclonal antibody recognizes APOBEC3G, APOBEC3A, and APOBEC3B due to unavoidable homology, in studies described in this disclosure, the antibody is only detecting APOBEC3G in the immunohistochemistry because APOBEC3 A is not expressed in T lymphocytes and, for reasons still under investigation, this monoclonal antibody does not recognize
- APOBEC3A is not expressed in most normal cell types, and it is only induced to detectable levels in interferon-activated myeloid lineage cells (Refsland et al. Nucleic Acids Res. 2010;38:4274-84; Koning et al. Journal of
- Kaplan-Meier curves were constructed by dividing specimens at the median expression level for each gene. Overall survival (OS) data was available for all 354 patients, while progression free survival (PFS) data was only available for 348 patients. P-values, hazard ratios and 95% confidence intervals were determined using Cox regression models on the continuous log2 -transformed expression, adjusting for stage and debulking status.
- Omnibus were compiled by the 2015 version of the Kaplan-Meier plotter database on a PostgreSQL server (Gyorffy et al. Endocr. Relat. Cancer. 2012;19: 197-208).
- GEO accession numbers were GSE14764, GSE15622, GSE18520, GSE19829, GSE23554, GSE26193, GSE26712, GSE27651, GSE30161, GSE3149, GSE51373, and GSE9891. All gene expression data were determined using only the HG-U133A, HG-U133 Plus 2.0, and HG-U133A 2.0 Affymetrix microarray platforms so that comparisons could be made between datasets.
- APOBEC3G expression was determined using the 204205_at probe.
- Grade 3 serous ovarian cancers were the only samples used in this analysis.
- OS data were available for 856 patients, while PFS data was available for 753 patients.
- Kaplan-Meier plots were constructed and p-values, hazard ratios and 95% confidence intervals were calculated using the Mantel-Cox log-rank test.
- APOBEC3G mRNA levels were measured by RTqPCR using an assay on demand (Hs00222415_ml, Applied Biosy stems) and three reference genes were measured with the primers listed in Table 3 and quantification using SYBR green. Relative APOBEC3G expression levels were determined by normalization to the average of 3 reference genes as described (Sieuwerts et al. Horm. Cancer. 2014;5:405-13). Kaplan-Meier curves were constructed by dividing specimens at the median expression level for each gene. P-values, hazard ratios and 95% confidence intervals were determined using the Mantel-Cox log-rank test.
- RNAseqV2 data were downloaded from TCGA in July 2015.
- APOBEC 3D, and CD20 mRNA levels were quantified using normalized read counts.
- r s and p- values for linear models of APOBEC versus immune-marker genes were calculated using
- APOBEC3G expression correlates with activated T lymphocyte infiltration in HGSOC
- APOBEC3G is expressed constitutively in many different cell lines and tissue types and is also known to be upregulated by HIV-1 infection of primary T lymphocytes, which is one of many distinct mechanisms of immune cell activation. Although virus infection is unlikely to be part of the etiology of ovarian cancer, the presence of activated immune cells (infiltrate) in HGSOC is known to correlate with better overall outcomes, most likely due to strong anti-tumor immune responses (Knutson et al. Cancer Immunol Immunother.
- RNA was prepared from fresh frozen HGSOC tissues, and a previously validated, highly specific RT-qPCR assay was used to assess APOBEC3G mRNA levels (Refsland et al. Nucleic Acids Res. 2010;38:4274-84). In parallel, expression of the related gene
- APOBEC3B which has been implicated in ovarian cancer genome mutagenesis (Leonard et al. Cancer Research. 2013;73 :7222-31), was assayed.
- the mRNA levels of several established T cell markers were quantified, including CD3D (total T cells), CD4 (helper T cells), CD8A (cytotoxic T cells), GZMB (activated cytotoxic T cells), PRF1
- APOBEC3G expression showed a similar positive correlation with CD8A and CD4, suggesting that AP0BEC3G is expressed in both the cytotoxic and helper T cell subsets (FIG. IB vs. 1C).
- This result was corroborated by similarly strong positive correlations between APOBEC3G and two markers of CTL activation, GZMB and PRF1 (FIG. ID and IE).
- GZMB and PRF1 FIG. ID and PRF1
- weaker and less significant correlations were found between APOBEC3B and the expression of any of these T cell genes (FIG. 1G-L). This result was expected, however, because prior studies have indicated that APOBEC3B is only expressed at low levels in normal tissues and upregulated in tumor cells.
- APOBEC3G expression (representative image in FIG. 2E).
- two tumors showed extensive T lymphocyte infiltration (representative images for CD3, CD4 and CD8 in FIG. 2G-2I, 2L-2N, and 2Q-2S).
- FIG. 2 J, 20, and 2T there was a strong colocalization between these markers and the expression of APOBEC3G in these tumors.
- FIG. 2 J, 20, and 2T different regions of a single HGSOC can be heterogenous, with one region showing dense clusters of APOBEC3G-high infiltrating T cells and another showing a more dispersed distribution (compare FIG. 2F-J and 2K-0).
- APOBEC3G is a candidate biomarker for improved HGSOC patient outcomes
- Kaplan-Meier plotter (kmplot.com) was used to generate a validation cohort using HGSOC data from TCGA and GEO.
- HGSOC specimens from a Dutch cohort consisting of 88 patient samples were analyzed.
- APOBEC3G expression was quantified using an independent RT-qPCR strategy (housekeeping gene primer sequences in Table 4 and see Materials and Methods for details). Again, APOBEC3G expression levels associated with improved OS and more weakly with PFS (FIG. 6).
- APOBEC3B has been implicated recently as an endogenous mutagen in several cancers, including ovarian cancer. Moreover, its overexpression has been linked to poor patient outcomes in multiple cancer types. Using the Mayo cohort gene expression data and clinical information from above, the effect of APOBEC3B on patient outcomes in HGSOC was examined. In contrast to prior reports for other cancers, the Mayo cohort showed a trend toward high APOBEC3B and improved, rather than worsened, PFS and OS outcomes, although these relationships were less significant statistically that those for APOBEC3G
- APOBEC expression correlates with immune cell markers in multiple human cancers
- RNAseq data from TCGA were analyzed to determine if correlations exist between expression of APOBEC genes and immune cell markers.
- the TCGA had RNAseq data available for 7,861 samples spanning 22 different tumor types (details in Table 5).
- expression of each APOBEC family member was quantified and correlated with the T cell marker CD3D (FIG. 4 top heatmap).
- Hierarchical clustering was also performed to elucidate similar correlation patterns between cancer types (FIG. 4).
- APOBEC3H also correlated significantly with CD3D across multiple cancer types.
- APOBEC3F also known to be expressed broadly and in T cells, did not correlate as strongly.
- CD20 a well-known marker for B cells (FIG. 4 bottom heatmap).
- the expression of the antibody diversification gene, AID was the only APOBEC family member that significantly correlated with CD20 in a majority of cancer types (FIG. 4).
- CD8A NM 001768 tcatggccttaccagtgacc (SEQ ID NO:5) aggttccaggtccgatcc (SEQ ID NO:17) UPL51
Abstract
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WO2008136852A2 (en) * | 2006-11-01 | 2008-11-13 | University Of Rochester | Methods and compositions related to the structure and function of apobec3g |
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US20120252026A1 (en) * | 2011-04-01 | 2012-10-04 | Harris Reuben S | Cancer biomarker, diagnostic methods, and assay reagents |
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